Background Ovarian cancer (OC) is the most lethal gynecologic malignancy, with disease progression critically shaped by the tumor microenvironment. Ascitic fluid constitutes a complex cellular and soluble niche that promotes tumor growth and immune evasion; however, the innate lymphoid landscape within OC ascites remains poorly characterized.

Methods We performed single-cell RNA sequencing of natural killer (NK) cells isolated from OC ascites to resolve innate immune heterogeneity and differentiation states. Functional assays assessed NK cell cytotoxicity, degranulation, and receptor expression following exposure to ascites, with or without transforming growth factor-β (TGF-β) blockade. Proteomic profiling was used to define the ascites cytokine milieu.

Results We identified eight distinct NK/innate lymphoid subclusters spanning cytotoxic, precursor, early-like, tolerant/immunoregulatory, regulatory, proinflammatory, and innate lymphoid cell (ILC) states. OC ascites were characterized by depletion of cytotoxic and precursor NK cells and enrichment of early-like, tolerant, regulatory, and proinflammatory populations. Pseudotime trajectory analysis revealed impaired maturation toward terminally differentiated cytotoxic NK cells. Notably, a functionally exhausted innate lymphoid cell 2 (ILC2) population accumulated in ascites and was associated with poor progression-free survival. Exposure of healthy donor NK cells to ascites impaired degranulation and killing of OVCAR-4 cells, reduced activating receptor expression (NKp30, DNAM-1), increased inhibitory receptor expression (PD-1, LAG-3, TIGIT), and induced a shift toward CD56^highCD16^low NK cells. Proteomic profiling revealed an immunosuppressive, type-2–polarized cytokine milieu enriched in TGF-β and ILC2-promoting mediators. Pharmacological TGF-β blockade partially restored NKp30/DNAM-1–dependent NK cell cytotoxicity.

Conclusions OC ascites establishes a type-2–skewed immunoregulatory niche that coordinately drives NK cell dysfunction and ILC2 accumulation. These findings provide a systems-level framework linking ascites-derived cues to innate immune remodeling and identify actionable pathways for restoring antitumor immunity in ovarian cancer.